beta(1)-Adrenergic receptor vs adenylyl cyclase 6 expression in cardiac myocytes: differences in transgene localization and intracellular signaling.

Adenylyl cyclase type 6 (AC6) and the beta(1) adrenergic receptor (beta(1)AR) are pivotal proteins in transmembrane betaAR-signaling in cardiac myocytes. Increased expression of AC6 has beneficial effects on the heart, but increased beta(1)AR expression has marked deleterious effects. Why do these two elements of the betaAR pathway have such different effects? Using adenovirus-mediated gene transfer of the two transgenes in neonatal rat cardiac myocytes, we assessed cellular distribution and performed selected biochemical assays.

Activation of PH-domain leucine-rich protein phosphatase 2 (PHLPP2) by agonist stimulation in cardiac myocytes expressing adenylyl cyclase type 6.

The Ser/Thr-specific phosphatase PHLPP (pleckstrin homology domain leucine-rich repeat protein phosphatase) regulates the amplitude and duration of agonist-evoked Akt signaling by dephosphorylating the hydrophobic motif (Ser473) of Akt, therefore inactivating Akt. We recently reported that gene transfer of adenylyl cyclase type 6 (AC6) into neonatal rat cardiac myocytes was associated with increased Akt phosphorylation and activity. To determine the underlying mechanisms for AC6-associated increase in Akt activation, we determined how AC6 gene transfer regulated the activity of PHLPP2 (one of the three PHLPP family phosphatases) in neonatal rat cardiac myocytes.

Adenylyl cyclase type VI increases Akt activity and phospholamban phosphorylation in cardiac myocytes.

Increased expression of adenylyl cyclase VI has beneficial effects on the heart, but strategies that increase cAMP production in cardiac myocytes usually are harmful. Might adenylyl cyclase VI have beneficial effects unrelated to increased beta-adrenergic receptor-mediated signaling? We previously reported that adenylyl cyclase VI reduces cardiac phospholamban expression. Our focus in the current studies is how adenylyl cyclase VI influences phospholamban phosphorylation.

Cardiac-directed expression of adenylyl cyclase VI facilitates atrioventricular nodal conduction.

Objectives: The purpose of this study was to test the hypothesis that cardiac-directed expression of adenylyl cyclase VI (AC(VI)) facilitates atrioventricular (AV) nodal conduction.

Background: Cardiac-directed expression of AC(VI), unlike other strategies to increase cyclic adenosine monophosphate generation, reduces mortality in murine cardiomyopathy. Recent reports suggest that AC(VI) expression may also protect against lethal bradycardia.

Quantifying estrogen and progesterone receptor expression in breast cancer by digital imaging.

Developments in digital imaging and fluorescent microscopy provide a new method and opportunities for quantification of protein expression in human tissue. Archived collections of paraffin-embedded tumors can be used to study the relationship between quantitative differences in protein expression in tumors and patient outcome. In this report we describe the use of a DeltaVision Restoration deconvolution microscope, combined with fluorescent immunohistochemistry, to obtain reproducible and quantitative estimates of protein expression in a formalin-fixed paraffin-embedded tissue.

Rho-mediated cytoskeletal rearrangement in response to LPA is functionally antagonized by Rac1 and PIP2.

G-protein-coupled receptors signal through Rho to induce actin cytoskeletal rearrangement. We previously demonstrated that thrombin stimulates Rho-dependent process retraction and rounding of 1321N1 astrocytoma cells. Surprisingly, while lysophosphatidic acid (LPA) activated RhoA in 1321N1 cells, it failed to produce cell rounding.

Targeted deletion of protein kinase C lambda reveals a distribution of functions between the two atypical protein kinase C isoforms.

Protein kinase C lambda (PKClambda) is an atypical member of the PKC family of serine/threonine kinases with high similarity to the other atypical family member, PKCzeta. This similarity has made it difficult to determine specific roles for the individual atypical isoforms. Both PKClambda and PKCzeta have been implicated in the signal transduction, initiated by mediators of innate immunity, that culminates in the activation of MAPKs and NF-kappaB.

Adenylyl cyclase type VI gene transfer reduces phospholamban expression in cardiac myocytes via activating transcription factor 3.

Cardiac-directed expression of adenylyl cyclase type VI (AC(VI)) increases stimulated cAMP production, improves heart function, and increases survival in cardiomyopathy. In contrast, pharmacological agents that increase intracellular levels of cAMP have detrimental effects on cardiac function and survival. We wondered whether effects that are independent of cAMP might be responsible for these salutary outcomes associated with AC(VI) expression.

Nkx2-5 pathways and congenital heart disease; loss of ventricular myocyte lineage specification leads to progressive cardiomyopathy and complete heart block.

Human mutations in Nkx2-5 lead to progressive cardiomyopathy and conduction defects via unknown mechanisms. To define these pathways, we generated mice with a ventricular-restricted knockout of Nkx2-5, which display no structural defects but have progressive complete heart block, and massive trabecular muscle overgrowth found in some patients with Nkx2-5 mutations. At birth, mutant mice display a hypoplastic atrioventricular (AV) node and then develop selective dropout of these conduction cells.

Indirect intracoronary delivery of adenovirus encoding adenylyl cyclase increases left ventricular contractile function in mice.

We performed indirect intracoronary delivery of adenovirus vectors in mice and explored techniques including hypothermia and pharmacological means to increase cardiac gene transfer. Mice were maintained in a normothermic state or cooled to 25 degrees C. The aorta or both the pulmonary artery and aorta were clamped while a needle was advanced into the left ventricular cavity to deliver adenovirus vectors encoding enhanced green fluorescent protein (EGFP) or murine adenylyl cyclase type VI (AC(VI)) with saline, sodium nitroprusside, acetylcholine, or serotonin.

Actin polymerization in macrophages in response to oxidized LDL and apoptotic cells: role of 12/15-lipoxygenase and phosphoinositide 3-kinase.

Formation of filamentous F-actin drives many cellular processes, including phagocytosis and cell spreading. We have recently reported that mouse macrophage 12/15-lipoxygenase (12/15-LO) activity promotes F-actin formation in filopodia during phagocytosis of apoptotic cells. Oxidized low-density lipoprotein (OxLDL) also stimulates robust F-actin formation and spreading of macrophages.

Association of ataxia telangiectasia mutated (ATM) gene mutation/deletion with rhabdomyosarcoma.

Rhabdomyosarcoma is a common malignancy in children. There are two major types of rhabdomyosarcomas, the embryonal and the alveolar, differing in cytogenetic and morphologic features. The alveolar type of rhabdomyosarcoma is frequently associated with chromosome translocation t(2; 13) and poor clinical prognosis.

Minimally modified LDL binds to CD14, induces macrophage spreading via TLR4/MD-2, and inhibits phagocytosis of apoptotic cells.

Minimally modified low density lipoprotein (mmLDL) is a pro-inflammatory and pro-atherogenic lipoprotein that, unlike profoundly oxidized LDL (OxLDL), is not recognized by scavenger receptors and thus does not have enhanced uptake by macrophages. However, here we demonstrate that mmLDL (as well as OxLDL) induces actin polymerization and spreading of macrophages, which results in such pro-atherogenic consequences as inhibition of phagocytosis of apoptotic cells but enhancement of OxLDL uptake. We also demonstrate for the first time that the lipopolysaccharide receptor, CD14, and toll-like receptor-4/MD-2 are involved in these mmLDL effects.